P1485: PKM AND PKR EXPRESSION DURING HEMATOPOIESIS AND ERYTHROPOIESIS
Authors: Erin Tsai, Leonardo Rivadeneyra, Saliha Yilmaz, Lenny Dang, Megan Wind-Rotolo
Journal: HemaSphere (2023)

Topic: 28. Enzymopathies, membranopathies and other anemias Background: Dysregulation of hematopoiesis and ineffective erythropoiesis are linked to many complications and comorbidities that are associated with serious disorders, such as thalassemia, myelodysplastic syndrome-associated anemia, sickle cell disease, and pyruvate kinase (PK) deficiency. A key enzyme in the glycolytic pathway, PK is needed for the production of adenosine triphosphate, which is essential for overall red blood cell health. The 4 main tissue-specific PK isoforms are encoded by 2 genes. The PKLR gene encodes the PKL and PKR isoforms through tissue-specific promoters, and the PKM gene encodes the PKM1 and PKM2 isoforms through alternative splicing. mRNA expression varies throughout the stages of normal hematopoiesis and erythropoiesis. In this study, transcriptomes from hematopoietic and erythroid progenitors were evaluated, and mRNA levels of PKL, PKR, PKM1, and PKM2 were measured at different stages of hematopoiesis and erythropoiesis. Aims: To understand relative expression and potential roles of PK isoforms during normal hematopoiesis and erythropoiesis. Methods: Two RNA-sequencing data sets were obtained from public functional genomics data repositories. (1) PRJEB19300/E-MTAB-5456 (CD34+, lineage-depleted umbilical cord blood from normal/healthy donors): transcriptomes of multipotent long-term hematopoietic stem cells (LT-HSC), multipotent progenitors (MPP), common myeloid progenitors (CMP), and megakaryocyte erythroid progenitors (MEP). (2) PRJNA475757/GSE115678 (in vitro cultured CD34+-derived erythroblasts): transcriptomes of CMP, colony-forming unit – erythroid (CFU-E), proerythroblasts (ProE), basophilic erythroblasts (BasoE), polychromatic erythroblasts (PolyE), orthochromatic erythroblasts (OrthoE), and reticulocytes (Retic). Raw sequencing data were processed using FastQC, Trimmomatic, Spliced Transcripts Alignment to a Reference (STAR), and RNA-Seq by Expectation-Maximization (RSEM). RSEM-normalized data were preprocessed to provide a transcripts-per-million (TPM) matrix. PKL, PKR, PKM1, and PKM2 expression data were visualized using RStudio. Results: In the early hematopoietic data set, PKR, PKL, and PKM1 transcripts had low to no detectable expression in any cell type. PKM2 was expressed (minimum, mean, maximum TPM) in LT-HSCs (37.3, 63.4, 120.3), MPPs (0.0, 47.4, 78.5), CMPs (67.3, 86.7, 113.4), and MEPs (45.5, 64.0, 101.3). In the erythroid progenitors’ data set, PKM1 transcripts were not detected, and PKL transcripts were detected at extremely low levels. PKR transcripts were detected in CFU-E (11.9, 42.8, 62.6), ProE1 (52.5, 72.8, 84.4), ProE2 (65.8, 75.5, 81.8), and BasoE (5.4, 14.1, 23.5) cell types. PKM2 was detected in CMPs (76.3, 291.4, 489.0), CFU-Es (38.5, 65.3, 86.8), and ProE1 (19.7, 22.1, 25.5). PKM2 and PKR expression during early hematopoiesis and erythropoiesis are illustrated in Figure 1. Summary/Conclusion: These analyses showed that transcripts of PKM1 and PKL were present at low-to-undetectable levels in hematopoietic and erythroid progenitors. PKM2 transcripts were present at early stages of hematopoiesis and were the dominant isoform compared with PKR. Importantly, PKM2 and PKR transcripts were both expressed in early erythropoiesis, with the level of PKM2 expression decreasing upon maturation. Similar studies of transcriptomes derived from patients with diseases that feature compromised erythrocyte maturation may inform effective pharmacotherapeutic approaches.Keywords: CD34+ cells, Pyruvate kinase, Hematopoiesis, Erythropoieisis